Isolation and characterisation of Arabidopsis mutants altered in the regulation of flavonoid biosynthetic genes by UV-B and blue light

Abstract

This thesis presents the application of a genetic approach to study aspects of the regulation of flavonoid biosynthesis. The gene encoding the first committed step in flavonoid biosynthesis is chalcone synthase (CHS). CHS is induced by a range of biotic and abiotic stimuli in Arabidopsis thaliana. Among these stimuli are various light qualities, including UV-B (280 320 nm) and blue light (390-500 nm). These light qualities each have the capacity to stimulate chalcone synthase gene expression, but when UV-B and blue light are used together to illuminate Arabidopsis, CHS expression is induced synergistically (i.e. to a greater than additive degree). As no genetic elements of the synergistic mechanism had been identified, a screen for mutants in UV-B and blue light synergistic induction of CHS in Arabidopsis thaliana was developed and carried out. A forward genetic approach was employed to identify mutants, using a transgenic line in which firefly luciferase (Liic) was expressed under the control of the CHS promoter. Luciferase can easily be visualised in plants, allowing mutants in the regulation of CHS to be selected. The screen resulted in the identification of a number of mutants. Among them was probably a novel uvr8 {ultra-violet resistant locus 8) allele. A second approach to study the genetic basis of flavonoid gene regulation was to further characterise a previously isolated mutant, icx2 (increased chalcone synthase expression 2). icx2 was originally identified as a mutant over-expressing CHS in the presence of UV-B. Previous characterisation had shown icx2 to over-express not only CHS, but also other genes of the flavonoid biosynthetic pathway in response to a range of light qualities. In addition, icx2 individuals had been shown to have reduced stature and deep purple leaves. The purple leaf phenotype resulted from hyper-accumulation of anthocyanins. The work detailed here further characterised the pleiotropic phenotype of icx2, identifying a new short root phenotype. The plant hormone auxin has an effect on root architecture and has its own function disrupted by flavonoids. Experiments were undertaken to assess the effect of flavonoid accumulation in icx2. The root phenotype was seen in individuals grown on medium containing sucrose and was shown to be independent of CHS over-expression or flavonoid accumulation. A screen for suppressor mutants of the icx2 mutation was carried out, which suggested the pleiotropic phenotypes of icx2 are monogenic. Map-based cloning of icx2 had been attempted before, but had been hindered by an unreliable phenotype. Efforts were made in this study to overcome the problems with the phenotype. In addition, mapping resources and data were produced